The major specific objectives of this proposal are: 1. to analyze the kinetics and mechanism of action of glyceraldehyde phosphate dehydrogenase using stopped flow as well as conventional kinetic techniques in order to determine how the structure of the coenzyme affects the intermediate steps of the enzyme mechanism; 2. to investigate the interaction of NADH with glyceraldehyde phosphate dehydrogenase both in its native form and as an acyl (or subsituted) enzyme in order to confirm or disprove the proposal that the reduced coenzyme alters the conformation and function of the acyl intermediate; 3. to examine further the unusual anti-cooperative and cooperative binding of pyridine nucleotides to different glyceraldehyde dehydrogenases in order to find out how these phenomena are related to alterations in structure or conformation of the proteins; and 4. to study the kinetic properties of glyceraldehyde phosphate dehydrogenase under approximately physiological conditions in vitro in order to determine the effects of high protein concentration and interactions with other enzymes on the activity of the dehydrogenase in vivo. The information gained in these investigations should be valuable in understanding how structure and function are related in this important enzyme.